Numerous methods have been developed for the incorporation of ligands into synthetic oligonucleotides using phosphoramidite chemistry. See S. L. Beaucage and P. I. Radhakrishnan, Tetrahedron 49:1925 (1993). One approach is based on the incorporation of an aliphatic amino group into synthetic oligonucleotides by use of an amino-modifier phosphoramidites or modified CPGs. See S. Agrawal et al., Nucleic Acids Res., 14: 6227 (1986). See P. S. Nelson et al., Nucleic Acids Res. 20:6253 (1992); P. S. Nelson et al., Nucleic Acids Res. 17:7179 (1989); P. S. Nelson et al., Nucleic Acids Res. 17: 7187 (1989). Such amino groups allow the post-synthetic addition of a variety of molecules including fluorescent markers, biotin, haptens or enzymes which can be used as hybridization probes for non-radioactive detection of specific target DNA/RNA or as sequencing primers. Amino-modified oligonucleotides have also been used for preparation of affinity matrices and as immobilized PCR primers.
One disadvantage of covalent labeling and immobilization of oligonucleotides is that the covalent bond is not selectively cleavable, and, therefore does not allow for the recovery/release of oligonucleotide or the removal of the label. In order to circumvent this problem, several approaches have been proposed based on chemical cleavage. These include the use of cleavable disulfide bonds or the introduction of acid cleavable linkers. See W Bannwarth and J. Wippler, Helv. Chim. Acta 73:1139 (1990); B. D. Gildea et al., Tetrahedron Lett. 31:7095 (1990); E.Leikauf et al., Tetrahedron 51:3793 (1995); J. Olejnik et al., Nucleic Acids Res. 24: 361 (1996). These approaches, however, require the application of chemical reagents that are not suitable for many applications. They also necessitate removal of the excess of cleaving reagents and result in residual modification of the oligonucleotide.